Phosphorylation of protein tyrosine residues is a cell's primary mechanism for propagating membrane receptor signals and for regulating cell growth and oncogenic transformation. A recently identified and growing family of protein tyrosine phosphatases, enzymes that specifically remove the phosphate moieties from phosphotyrosine- containing proteins, are essential for priming these signalling pathways and for controlling the levels of cell growth. Despite the centrality of these regulatory processes, no tertiary structures are known of any of the key enzymes. This proposal describes preliminary results and strategies to determine the X-ray crystal structure of a bacterial protein tyrosine phosphatase, with and without bound inhibitors and substrates, which will serve as a paradigm for understanding the enzyme's function and specificity. The pathogenic bacterium Yersinia is responsible for a range of human and rodent diseases from diarrhea to the bubonic plague. An essential determinant of its virulence is a secreted protein tyrosine phosphatase termed Yop51. Yop51 is likely a toxin which may enter host cells, interfere with immune cell phosphorylation levels and activation pathways, and allow Yersinia to subvert the host's immune system surveillance. The catalytic portion of this enzyme is highly homologous to human tyrosine phosphatases, has similar substrate specificity, and has an identical catalytic mechanism centered around an essential cysteine. Additionally, it can be expressed in large amounts for biochemical and structural studies. Large, well-diffracting crystals have been obtained of the apoenzyme. Data have been collected and a search for heavy atom derivatives is in progress. Diffracting crystals of the phosphatase complexed with the potent oxyanion inhibitor tungstate were also obtained and differ from the native. This structure may mimick the unique phosphocysteine enzyme transition state. In addition, procedures are described for crystallizing a phosphotyrosine peptide substrate bound to an enzyme variant incapable of substrate turnover. This will reveal how the enzyme catalyzes phosphate removal, why it is specific for phosphotyrosine, and how substrate-protein interactions define the enzyme's specificity.